52 Current Pharmaceutical Design, 2012, Vol. 18, No. 1 Ozawa et al. H3 C OH H 3C CH3 CH3 CH3 CH3 CH3 H3C CH3 HO CH 3Fig. (1). Chemical structure of lutein.C40H 56O2 . Lutein contains double bonds that scavenge ROS.Fig. (2). The human retina.(A) Fundus photograph. An arrow shows the center of the retina, where photoreceptor cells and light stimuli are concentrated. (B) Optical coherence tomogra-phy (OCT) image showing a cross section of the central part of the retina. NFL, nerve fiber layer; OPL, outer plexiform layer; ONL, outer nuclear layer (pho-toreceptor cell layer); OS, outer segment.those who took a large amount of lutein or zeaxanthin  and who 3. BIOLOGICAL EFFECTS AND UNDERLYING MOLECU-showed high levels of plasma carotenoid [13,14] but not of other LAR MECHANISMS OF LUTEIN IN MOUSE RETINALantioxidants, vitamin C, E, or selenium . DISEASE MODELS A study using donor eyes showed a negative association be-tween lutein content in the retina and AMD risk . Retinas from 1) Lutein’s Effects on Acute Innate Inflammation of the Retina56 donors with AMD and 56 controls were analyzed using high- To simplify the analysis of lutein’s biological actions in vivo,performance liquid chromatography. The levels of lutein and zeax- we first used an endotoxin-induced uveitis (EIU) mouse model,anthin were lower in the AMD retinas in both the central retina generated by the injection of lipopolysaccaride (LPS) .(62% of control), including the macula, and the peripheral retina(70-80% of control). Taken together, these observations indicate i) Retinal Neuronal Disorder During Inflammationthat lutein obtained from the diet accumulates in the retina and may In the EIU model, the innate immune system causes intraocularact locally to prevent disease. inflammation, which involves the neural retina [20-24]. In human In a second large-scale clinical study, AREDS2, the effects of uveitis cases, inflammation spreads to the retina as well as the uvealutein/zeaxanthin and/or docosahexaenoic/eicosapentaenoic acids in and causes visual dysfunction, although the underlying mechanismapproximately 4000 AMD patients are presently being tested for has long been obscure. In the EIU model mice, electroretinogramtheir possible use in preventive therapy. This possibility is based on (ERG), an objective method for measuring visual function in boththe involvement of oxidative stress-related conditions, such as humans and mice, shows a decrease in the a-wave amplitude, andsmoking and hypertension , in the risk for AMD, and the pro- photoreceptor cell dysfunction [20,21,23,24]. This effect involves aposed anti-oxidative action of lutein. However, the biological role reduction in rhodopsin protein, which is localized in the OS of pho-of lutein in the body, including the retina, has not yet been eluci- toreceptor cells and is indispensable for photo-transduction. Thisdated. The association between arthritis and AMD , and be- protein reduction results from excessive protein degradation by thetween the genetic abnormality of complement factor and AMD [17- ubiquitin-proteasome system (UPS), in which the protein-selective19], have also been reported, suggesting that inflammation is re- E3-ligase, ubiquitin-protein ligase E3 component n-recognin 1lated to AMD. However, an anti-inflammatory effect of lutein has (Ubr1) , is upregulated by STAT3 activation downstream ofbeen hardly documented. inflammatory signals, such as interleukin-6 (IL-6). The loss of rhodopsin shortens the OSs  and decreases light reception and The retina is part of the central nervous system, and thus regen- photoreceptor cell function. Under physiological conditions, sup-erates poorly, if at all. Therefore, it is essential to protect the retinal pressor of cytokine signaling 3 (SOCS3), which modulates acti-neurons from oxidative stress and inflammation. Many adult dis- vated STAT3 via a feedback loop, fine-tunes this signal [24,26].eases, including diabetes and AMD, involve oxidative stress and Once this feedback system is overwhelmed, however, the STAT3inflammation. To investigate lutein’s potential as a preventative activation and subsequent rhodopsin reduction are accelerated,therapy for these diseases, it is important to study its mechanisms of resulting in vision loss [23,24].action in vivo.
Lutein Protects Neurons Current Pharmaceutical Design, 2012, Vol. 18, No. 1 53ii) Lutein’s Effect During Inflammation Includes ROS Suppres- STAT3 activation, the direct pathway of STAT3 phosphorylationsion and activation by ROS 27 may be suppressed by lutein. Therefore, The UPS is also accelerated by protein modifications resulting there is a link between oxidative stress and inflammation, whichfrom oxidative stress. Therefore, if oxidative stress is involved in can be regulated by lutein; thus, lutein has an anti-inflammatoryinflammation and lutein has an anti-oxidative effect in vivo, lutein effect (Fig. 4). This scenario is supported by previous reports show-might protect visual function by suppressing inflammation-related ing that lutein reduces the intraocular infiltration of inflammatoryprotein degradation. cells in models of EIU  and laser-induced choroidal neovascu- larization . To test this hypothesis, we pre-treated EIU model mice withlutein, and examined whether the rhodopsin protein level, OS STAT3 activation and its related pathological changes in thelength, and visual function (photoreceptor cell function) were pre- retina of the EIU model are also suppressed by angiotensin II type 1served during inflammation . Interestingly, oxidative stress was receptor (AT1R) blockers . This is not only because AT1Rinduced in the retina during inflammation, and lipid peroxidation blockers suppress STAT3 activation downstream of AT1R, but alsowas observed in the plasma membrane of the OSs, supporting the because they suppress the ROS generation by NADPH oxidaseidea that rhodopsin, a transmembrane protein, was modified by the downstream of AT1R signaling.inflammation. These effects were successfully suppressed by lutein 2) Lutein’s Effects on Diabetic Retinopathy(Fig. 3), indicating that lutein protects the function of the retinalneural tissue by reducing oxidative stress and protein loss. Consis- Although chronic neurodegeneration is well known to occur intently, lutein preserved a-wave amplitude in ERG, indicating that diabetes, there is no established therapy to prevent it. Given thatlutein protects visual function during inflammation. diabetes involves oxidative stress, a constant intake of the anti- oxidant, lutein, might help to prevent this complication in the retina.iii) Lutein Suppresses Inflammatory Signaling i) Retinal Neurodegeneration in Diabetes Lutein’s effect on rhodopsin preservation is accompanied by asuppression of STAT3 activation. STAT3 is generally activated by In a mouse model of type 1 diabetes induced by injecting strep-inflammatory cytokines, one of which, IL-6 is regulated by acti- tozotocin (STZ), a compound that is toxic to the insulin-producingvated STAT3. Thus, once STAT3 activation exceeds a certain level, beta cells of the pancreas, impaired visual function is obvious onethe IL-6-STAT3 pathway enters a vicious cycle that exacerbates the month after the onset of diabetes . The ERG shows impairedpathological condition. However, lutein suppresses STAT3 activa- oscillatory potentials (OPs), which reflects inner retinal neurode-tion, thereby suppressing this vicious cycle, and efficiently prevent- generation. This change is also observed in humans from an earlying tissue damage. Regarding the mechanism for inhibiting the stage, before diabetes-induced microangiopathy is obvious [31,32]. EIU EIU Control Vehicle Lutein Control Vehicle Lutein B C E F G Oxidized BODIPY-C11 intensity oxidized/non-oxidized A ONL INL DHE GCL H I J OS ONL INL D K * * Fluorescence intensity 2 * * 2.5 2 1.5 1.5 1 1 0.5 0.5 0 0 Control Vehicle Lutein Control Vehicle Lutein EIU EIUFig (3). Lutein’s effect on oxidative stress in the inflamed retina.ROS in the retina were detected by dihydroethidium (DHE) (A-D) and BODIPY-C11 which indicates lipid peroxidation (E-K). Lutein administration sup-presses ROS in the retina. (Sasaki et al. Invest Ophthalmol Vis Sci 2009).
54 Current Pharmaceutical Design, 2012, Vol. 18, No. 1 Ozawa et al.The retinal neurodegeneration and visual dysfunction continue to ii) Lutein Protects Neuronal Function in the Diabetic Retinaprogress after microangiopathy has been made quiescent by laser Strikingly, diabetic mice constantly fed a lutein-supplementedtreatment or surgical intervention. This neuronal disorder is caused diet show preserved visual function as measured by ERG, indicat-by the diabetic and toxic microenvironment. ing the prevention of inner retinal damage . Lutein suppresses We showed that local AT1R signaling contributes to this retinal the generation of ROS in the diabetic retina without changing theneurodegeneration . We found that the angiotensin II signal is systemic blood glucose level.upregulated locally in the retina, and the suppression of neuronal In the retina of lutein-fed diabetic mice, ERK activation is sup-damage by AT1R blockers was not associated with a reduction in pressed and synaptophysin protein is preserved. Therefore, thesystemic blood glucose levels. Inner retinal layer involves a neu- chronic activation of ERK is also associated with oxidative stressronal synaptic network where primary integration of the visual in- and is affected by lutein’s anti-oxidative and anti-inflammatoryformation occurs via active neurotransmitter release. Synaptic vesi- actions, to protect neuronal activity. Moreover, lutein is reported tocles are indispensable for this biological activity, and synapto- bind to a protein, glutathione-S-transferase 1, in the monkey retina,physin, a synaptic-vesicle component, plays a key role in various which localizes to the synaptic region, OPL, in addition to the OSsneurodegenerative diseases, most likely through synaptic network of photoreceptor cells . This distribution is consistent with lu-abnormalities; genetically abnormal synaptophysin causes mental tein’s protection of retinal neurons.retardation , and abnormal forms of it are found in Alzheimer’sdisease and Parkinson’s disease . Lutein’s protection of synaptic vesicle protein affects more than the synaptic activity. Neuronal activity induces the translocation of We showed that the protein level of synaptophysin is also re- calcium ions, which needs to be regulated to maintain cellular sur-duced in the diabetic retina through the pro-inflammatory AT1R vival and induces the expression of a neuronal trophic factor, brain-signaling pathway  by activating Extracellular Signal-regulated derived neurotrophic factor (BDNF) [39,40]. BDNF, whose defi-Kinase (ERK). ERK can induce seven in absentia (Sina) , ciency is associated with a number of neurodegenerative disorderswhich mammalian homologues, the seven in absentia homologues (e.g. Huntington’s disease, Alzheimer’s disease, and Parkinson’s(Siahs), have turned out to be a synaptophysin-selective E3-ligase disease), is also downregulated in the diabetic retina . Impor-. The involvement of a common pathogenic pathway, i.e., pro- tantly, however, lutein attenuates this decrease, most likely throughtein degradation, in the diabetes and EIU models is interesting, and its protection of synaptic activity (Fig. 4). It has not been deter-consistent with previous findings that diabetic influence in the ret- mined whether BDNF can be modified or degraded by oxidativeina involves inflammation [21,37] and oxidative stress . Since stress.locally activated AT1R signaling can cause oxidative stress, wehypothesized that a constant intake of lutein might prevent the reti- It is anticipated that neurodegenerative diseases will be treatednal neurodegeneration induced by diabetes. using strategies designed to increase the BDNF level  —the administration of recombinant proteins or drugs that activate its Inflammation and Diabetes (pro-infalmmatory molecules Light Exposure e.g. IL-6, Angiotensin II Oxidative Stress Lutein Downstream signaling (e.g. STAT3, ERK) Degradation of DNA damage Functional Proteins Neuronal Dysfunction (e.g. Rhodopsin<photo-transduction, Synaptophysin<neuronal activity & Neuronal Death neurotrophic factors)Fig. (4). Model of the underlying mechanisms of lutein’s effect.Lutein suppresses oxidative stress in tissue. This suppresses the degradation of functional proteins and DNA damage caused by pro-inflammatory moleculesinduced in the tissue during inflammation or diabetes, or as a result of light exposure, preventing neuronal dysfunction and death. Lutein can absorb and blocklight.
Lutein Protects Neurons Current Pharmaceutical Design, 2012, Vol. 18, No. 1 55endogenous expression and gene therapy to relieve the symptoms of ERG = Electroretinogram;Alzheimer’s disease and Parkinson’s disease are now in trial. If the ERK = Extracellular Signal-regulated Kinase;BDNF reduction in these neurodegenerative diseases involves amechanism common to that of diabetic retinopathy, lutein admini- IL = Interleukin;stration might also be considered as a therapeutic strategy for these LPS = Lipopolysaccaride;neuronal diseases. ONL = Outer nuclear layer;iii) Lutein Promotes Neuronal Survival in the Diabetic Retina OPL = Outer plexiform layer; Reductions in the neuronal functional protein synaptophysin OSs = Outer segments;and in the neurotrophic factor BDNF are implicated in the neuronal UPS = Ubiquitin-proteasome systemdysfunction of diabetic model mice, as shown by ERG . 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